The Importance of Accurate Microorganism Identification in Microbial Challenge Tests of Membrane Filters. Part II. The Comparison of Hydrogenophaga pseudoflava ATTC 33668 and Curvibacter sp. ATCC 700892 by Microbial Challenge Tests with Membrane Filters.

The ASTM 838-05 standard describes a bacteria challenge test procedure based on Brevundimonas diminuta (ATCC 19146) to verify a 0.2 µm rated sterilizing-grade filter. For process validation procedures a correct identification of the challenge organism is essential. The test strain ATCC 700892 repeatedly used for microbial challenge tests was incorrectly named Hydrogenophaga pseudoflava but is phylogenetically linked to the genus Curvibacter, as shown in Part I of this series. Based on these studies the misconception was consolidated that Hydrogenophaga pseudoflava, a widely isolated microorganism also found in biopharmaceutical production settings, is able to penetrate 0.2 µm rated filters. Here we show that the bacteria challenge test results of the strains Curvibacter sp. ATCC 700892 and Hydrogenophaga pseudoflava ATCC 33668 are different. In previous challenge tests analytical filter membranes were used; these do not represent the process scenarios within the sterilizing filtration in industrial processes. To represent process systems, the study data presented were determined with 10" filter cartridge elements. The strain Hydrogenophaga pseudoflava ATCC 33668 is completely retained by 0.2 µm and 0.1 µm rated filters. Depending on the different 0.2 µm filter material there are different retention rates of the strain Curvibacter sp. ATCC 700892; only the 0.1 µm rated filters showed consistent complete retention. However, up to date the genus Curvibacter seems to be of low relevance within biopharmaceutical production settings. LAY ABSTRACT: Bacteria challenge tests are used to determine the retention performance of sterilizing-grade filters. The model organism used for bacteria challenge tests and the verification of a 0.2 µm rated sterilizing-grade filter is Brevundimonas diminuta. In previous studies another proposed, model organism used for challenge tests was incorrectly labelled as Hydrogenophaga pseudoflava. Given the predefined retention characteristics, this mislabelled organism was recommended to be included in bacteria challenge studies. However, the herein presented testing demonstrated that the organism is actually phylogenetically affiliated to the genus Curvibacter and not to the strain Hydrogenophaga pseudoflava ATCC 33668. In this report, we demonstrate that the retention of the strain Hydrogenophaga pseudoflava ATCC 33668 with 0.1 µm and 0.2 µm rated filters is comparable to the retention of Brevundimonas diminuta ATCC 19146.

The importance of accurate microorganism identification in microbial challenge tests of membrane filters--part I.

Microbial challenge testing is a common procedure to determine the retention efficiency, performance, and validity of a sterilizing-grade filter. The ASTM 838-05 standard describes a bacteria challenge test procedure based on Brevundimonas diminuta (ATCC 19146), routinely used to verify a 0.2 µm rated sterilizing-grade filter. Process validation procedures most often also utilize B. diminuta (ATCC 19146), but instead of the standard procedures and fluids, process, and product parameters are employed to determine whether these parameters influence the retentivity of the filter or changes to the challenge organism, which might result in the penetration of the filter. In certain instances, the native bioburden within the drug manufacturing process is used to perform such process validation challenge tests. Filter penetrations can happen and cause concern; therefore, it is essential to identify the organism species with accuracy to avoid unnecessary confusion. This paper and its follow-up will describe such imprecision and the resulting misconceptions. It will clarify past determinations and put perspective on the findings. LAY ABSTRACT: Sterilizing-grade filters are used to remove microorganisms from biopharmaceutical solutions. To determine the retention performance of such filters, bacteria challenge tests are utilized, often with a standard challenge organism (Brevundimonas diminuta), in instances with native bioburden. The accuracy of the microorganism identification is of importance to avoid flawed results and misinterpretation of the filter's performance.

Validation of sterilizing grade filtration.

Validation consideration of sterilizing grade filters, namely 0.2 micron, changed when FDA voiced concerns about the validity of Bacterial Challenge tests performed in the past. Such validation exercises are nowadays considered to be filter qualification. Filter validation requires more thorough analysis, especially Bacterial Challenge testing with the actual drug product under process conditions. To do so, viability testing is a necessity to determine the Bacterial Challenge test methodology. Additionally to these two compulsory tests, other evaluations like extractable, adsorption and chemical compatibility tests should be considered. PDA Technical Report # 26, Sterilizing Filtration of Liquids, describes all parameters and aspects required for the comprehensive validation of filters. The report is a most helpful tool for validation of liquid filters used in the biopharmaceutical industry. It sets the cornerstones of validation requirements and other filtration considerations.

Use of the radial forearm microvascular free-flap graft for cranial base reconstruction.

OBJECT: Reconstruction of the cranial base after resection of complex lesions requires creation of both a vascularized barrier to cerebrospinal fluid (CSF) leakage and tailored filling of operative defects. The authors describe the use of radial forearm microvascular free-flap grafts to reconstruct skull base lesions, to fill small tissue defects, and to provide an excellent barrier against CSF leakage. METHODS: Ten patients underwent 11 skull base procedures including placement of microvascular free-flap grafts harvested from the forearm and featuring the radial artery and its accompanying venae comitantes. Operations included six craniofacial, three lateral skull base, and two transoral procedures for various diseases. Excellent results were obtained, with no persistent CSF leaks, no flap failures, and no operative infections. One temporary CSF leak was easily repaired with flap repositioning, and at one flap donor site minor wound breakdown was observed. One patient underwent a second procedure for tumor recurrence and CSF leakage at a site distant from the original operation. CONCLUSIONS: Microvascular free tissue transfer reconstruction of skull base defects by using the radial forearm flap provides a safe, reliable, low-morbidity method for reconstructing the skull base and is ideally suited to "low-volume" defects.

The pressure hold/drop integrity test; its correlation to diffusive flow.

The pressure-drop/hold procedure enables the diffusive flow integrity testing of filters to be performed without breaching the system downstream of the filter. It is not necessary to measure volumetrically the diffused gas on the downstream side of the filter. By means of pressure transducers the pressure loss is determined upstream; thus eliminating the threat of sepsis due to down-stream invasions. The pressure decay exercise can be used to characterize the various filter types. A constancy of filter manufacture is required for a given filter type. Unless the pressure drop exceeds the value established as the maximum allowable decay, the filter is judged to be integral. It qualifies as a sterilizing grade filter. Excessive pressure decays will also eventuate from leaks, as from improperly sealed housings. Performed prior to the filtration, the procedure serves to eliminate the wasteful use of an imperfect system, whether caused by faulty sealing, incorrect filter type or flawed filters. Where leaks are detected, the filter can be reexamined for its integrity. To enable the pressure-drop procedure to serve as an integrity test, the measured pressure decays require being correlated with organism retention data. This is made possible by the arithmetic conversion of the pressure decay curve into the conventional diffusive airflow curve established to have such a correlation. The transformation of the pressure-drop curve into the differential airflow plot is automatically performed by certain of the automated integrity test machines. These devices, utilizing pressure transducers, are capable of measuring small pressure drops with requisite sensitivity; gauges commonly are not. Moreover, as previously stated, the measurements are advantageously made on the upside of the filter. The use of automated test machines is, therefore, recommended for the performance of the pressure hold/drop integrity test in furtherance of the practice of filter integrity testing.

Bacterial cell size and surface charge characteristics relevant to filter validation studies.

There are two recognized mechanisms whereby organisms are retained by liquid filters; namely, sieve-retention and adsorption. The efficiency of each may be influenced by the organism, suspending milieu, and by the filtration conditions. Validations of sterilizing filtrations require the use of organism suspensions in product-specific media. However, where the product is bactericidal to the challenge organism(s), surrogate solutions may be required. The ideal surrogate solution would minimize adsorptive retention, ensuring that the sterilizing action of the filter under consideration is the consequence of sieve-retention. This review explores the impact that various physicochemical factors may have on bacterial cell size and cell surface characteristics. An understanding of interactions among challenge bacteria, suspending fluid, and filter medium is essential for the development of surrogate solutions that provide a "worst case" mileu for filter validation studies or a "placebo," non-inhibitory challenge solution.

A safe, reliable method for skin-graft coverage of the radial forearm donor site.

Delayed donor-site healing remains one of the most significant disadvantages of the radial forearm free flap. In an effort to decrease morbidity at the donor site, the authors adopted a closure technique that utilized the flexor digitorum sublimis (FDS) and flexor pollicis longus (FPL) muscle bellies to cover the flexor carpi radialis (FCR) tendon prior to placement of a split-thickness skin graft. While this approach eliminated tendon exposure, two patients with postoperative median-nerve compression forced a modification of this technique. The authors now detach the radial attachment of the FDS muscle and mobilize the median nerve away from the underside of the muscle, to prevent kinking of the nerve when the FDS and FPL muscle bellies are sewn together. With these modifications, the technique retains its efficacy, but with an improved margin of safety for the median nerve.

The need for rigid fixation of combined parasymphyseal and bilateral subcondylar fractures.

Both miniplate and larger fracture plate fixation techniques have been utilized successfully in the treatment of mandibular fractures. Parasymphyseal fractures which occur in conjunction with bilateral subcondylar fractures, however, represent a special fracture pattern that is not well managed with miniplate fixation. Miniplates do not have the tensile strength to resist the strong, muscular-deforming forces that act on these fractures. This fracture pattern is best managed with anatomic symphyseal reduction and internal fixation with a 2.7 mm mandibular fracture plate and a 2.0 mm tension band plate. The subcondylar fractures can then be managed with either intermaxillary fixation or open reduction. This approach provides good functional results and can prevent late complications.

Diffusive airflow integrity testing.

The more certain implications to retention based upon definition of the largest pores present in a filter may be had where bubble point testing is possible. Where diffusive airflows interfere with bubble point determinations, the diffusive airflow measurements themselves, correlated to organism retentions, serve as integrity tests. Diffusive airflow measurements have their advantage in addition to serving as indicators of filter integrity. They can more precisely reveal filter incompatibilities. They can be used to gauge the completeness of a filter's wettability, and they are more revealing of a filter's clean water flow properties. In almost all its applications, however, diffusive airflow integrity testing requires defining the diffusive airflow curve by way of multipoint measurements using the liquid product in question. This is especially true in filter validation contexts where product-specific testing is necessitated. Once the full diffusional airflow curve has been elucidated, single-point diffusional flow testing is warranted, as in production integrity testing settings. Single point diffusive flow testing can be used as a standard integrity test method for use in the pharmaceutical production environment.